Pseudorabies virus protein

ABSTRACT

The present invention provides recombinant DNA molecules comprising a sequence encoding a pseudorabies virus (PRV) glycoprotein selected from the group consisting of gI, gp50, and gp63, host cells transformed by said recombinant DNA molecule sequences, the gI, gp50 and gp63 polypeptides. The present invention also provides subunit vaccines for PRV, methods for protecting animals against PRV infection and methods for distinguishing between infected and vaccinated animals.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of Ser. No. 100,817, filedJun. 29, 1987, now abandoned, which represented the national phase ofinternational PCT/US86/01761, filed Aug. 28, 1986, which was acontinuation-in-part of Ser. No. 886,260, filed Jul. 16, 1986, nowabandoned, which was a continuation-in-part of Ser. No. 784,787 (filedOct. 4, 1985); Ser. No. 801,799 (filed Nov. 26, 1985), and Ser. No.844,113 (filed Mar. 26, 1986), all now abandoned.

FIELD OF INVENTION

This invention relates to DNA sequences encoding pseudorabies virusglycoproteins and polypeptides related thereto. These DNA sequences areuseful for screening animals to determine whether they are infected withPRV and also for expressing the glycoproteins encoded thereby.

BACKGROUND OF THE INVENTION

Pseudorabies virus (PRV) is a disease which infects many species ofanimals worldwide. PRV infections are variously called infectious Bulbarparalysis, Aujeszky's disease, and mad itch. Infections are known inimportant domestic animals such as swine, cattle, dogs, cats, sheep,rats and mink. The host range is very broad and includes most mammalsand, experimentally at least, many kinds of birds (for a detailed listof hosts, see D. P. Gustafson, "Pseudorabies", in Diseases of Swine, 5thed., A. D. Leman et al., eds., (1981)). For most infected animals thedisease is fatal. Adult swine and possibly rats, however, are not killedby the disease and are therefore carriers.

Populations of swine are particularly susceptible to PRV. Although theadult swine rarely show symptoms or die from the disease, piglets becomeacutely ill when infected and death usually ensues in 24 to 48 hoursoften without specific clinical signs (T. C. Jones and R. D. Hunt,Veterinary Pathology, 5th ed., Lea & Febiger (1983)).

PRV vaccines have been produced by a variety of techniques andvaccination in endemic areas of Europe has been practiced for more than15 years. Losses have been reduced by vaccination, but vaccination hasmaintained the virus in the environment. No vaccine has been producedthat will prevent infection. Vaccinated animals that are exposed tovirulent virus survive the infection and then shed more virulent virus.Vaccinated animals may therefore harbor a latent infection that canflare up again. (See, D. P. Gustafson, supra).

Live attenuated and inactivated vaccines for PRV are availablecommercially in the United States and have been approved by the USDA(See, C. E. Aronson, ed., Veternary Pharmaceuticals & Bioligicals,(1983)).

Because adult swine are carriers of PRY, many states have institutedscreening programs to detect infected animals. DNA/DNA hybridization canbe used to diagnose actively infected animals utilizing the DNA sequenceof the instant invention. Some of the PRV glycoproteins of the presentinvention are also useful in producing diagnostics for PRV infectionsand also to produce vaccines against PRV.

PRV is a herpesvirus. The herpesviruses generally are among the mostcomplex of animal viruses. Their genomes encode at least 50 virusspecific proteins and contain upwards of 150,000 nucleotides. Among themost immunologically reactive proteins of herpesviruses are theglycoproteins found, among other places, in virion membranes and themembranes of infected cells. The literature on PRV glycoproteins refersto at least four viral glycoproteins (T. Ben-Porat and A. S. Kaplan,Virology, 41, pp. 265-73 (1970); A. S. Kaplan and T. Ben-Porat, Proc.Natl. Acad. Sci. U.S.A., 66, pp. 799-806 (1970)).

INFORMATION DISCLOSURE

M. W. Wathen and L. K. Wathen, J. Virol., 51, pp. 57-62 (1984) refer toa PRV containing a mutation in a viral glycoprotein (gp50) and a methodfor selecting the mutant utilizing neutralizing monoclonal antibodydirected against gp50. Wathen and Wathen also indicate that a monoclonalantibody directed against gp50 is a strong neutralizer of PRV, with orwithout the aid of complement, and that polyvalent immune serum ishighly reactive against gp50, therefore concluding that gp50 may be oneof the important PRV immunogens. On the other hand, it has been reportedthat monoclonal antibodies that react with the 98,000 MW envelopeglycoprotein neutralize PRV infectivity but that monoclonal antibodiesdirected against some of the other membrane glycoproteins have verylittle neutralizing activity (H. Hampl, et al., J. Virol., 52, pp.583-90 (1984); and T. Ben-Porat and A. S. Kaplan, "Molecular Biology ofPseudorabies Virus", in B. Roizman ed., The Herpesviruses, 3, pp. 105-73(1984)).

L. M. K. Wathen, et al., Virus Research, 4, pp. 19-29 (1985) refer tothe production and characterization of monoclonal antibodies directedagainst PRV glycoproteins identified as gp50 and gp83 and their use forpassively immunizing mice against PRV infection.

A. K. Robbins, et al., "Localization of a Pseudorabies VirusGlycoprotein Gene Using an E. coli Expression Plasmid Library", inHerpesvirus, pp. 551-61 (1984), refer to the construction of a libraryof E. coli plasmids containing PRV DNA. They also refer to theidentification of a PRV gene that encodes glycoproteins of 74,000 and92,000 MW. They do not refer to the glycoproteins of the instantinvention.

A. K. Robbins, et al., European patent application No. 85400704.4(publication No. 0 162 738) refers to the isolation, cloning andexpression of PRV glycoproteins identified as gII and gIII. They do notrefer to the PRV glycoproteins of the instant invention.

T. C. Mettenletter, et al., "Mapping of the Structural Gene ofPseudorabies Virus Glycoprotein A and Identification of TwoNon-Glycosylated Precursor Polypeptides", J. Virol., 53, pp. 52-57(1985), refer to the mapping of the coding region of glycoprotein gA(which they equate with gI) to the BamHI 7 fragment of PRV DNA. Theyalso state that the BamHI 7 fragment codes for at least three otherviral proteins of 65K, 60K, and 40K MW. They do not disclose or suggestthe DNA sequence encoding the glycoproteins of the instant invention orthe production of such polypeptides by recombinant DNA methods.

B. Lomniczi, et al., "Deletions in the Genomes of Pseudorabies VirusVaccine Strains and Existence of Four Isomers of the Genomes", J.Virol., 49, pp. 970-79 (1984), refer to PRV vaccine strains that havedeletions in the unique short sequence between 0.855 and 0.882 mapunits. This is in the vicinity of the gI gene. T. C. Mettenleiter, etal., "Pseudorabies Virus Avirulent Strains Fail to Express a MajorGlycoprotein", J. Virol., 56, pp. 307-11 (1985), demonstrated that threecommercial PRV vaccine strains lack glycoprotein gI. We have also foundrecently that the Bartha vaccine strain contains a deletion for most ofthe gp63 gene.

T. J. Rea et al., J. Virol., 54, pp. 21-29 (1985), refers to the mappingand the sequencing of the gene for the PRV glycoprotein that accumulatesin the medium of infected cells (gX). Included among the flankingsequences of the gX gene shown therein is a small portion of the gp50sequence, specifically beginning at base 1682 of FIG. 6 therein.However, this sequence was not identified as the Kp50 sequence.Furthermore, there are errors in the sequence published by Rea et al.Bases 1586 and 1603 should be deleted. Bases should be inserted betweenbases 1708 and 1709, bases 1737 and 1738, bases 1743 and 1744 and bases1753 and 1754. The consequence of these errors in the published partialsequence for gp50 is a frameshift. Translation of the open reading framebeginning at the AUG start site would give an incorrect amino acidsequence for the gp50 glycoprotein.

European published patent application 0 133 200 refers to a diagnosticantigenic factor to be used together with certain lectin-bound PRVglycoprotein subunit vaccines to distinguish carriers and noncarriers ofPRV.

SUMMARY OF INVENTION

The present invention provides recombinant DNA molecules comprising DNAsequences encoding polypeptides displaying PRV glycoproteinantigenicity.

More particularly, the present invention provides host cells transformedwith recombinant DNA molecules comprising the DNA sequences set forth inCharts A, B, and C, and fragments thereof.

The present invention also provides polypeptides expressed by hoststransformed with recombinant DNA molecules comprising DNA sequences ofthe formulas set forth in Charts A, B, and C, and immunologicallyfunctional equivalents and immunogenic fragments and derivatives of thepolypeptides.

More particularly, the present invention provides polypeptides havingthe formulas set forth in Charts A, B, and C, immunogenic fragmentsthereof and immunologically functional equivalents thereof.

The present invention also provides recombinant DNA molecules comprisingthe DNA sequences encoding pseudorabies virus glycoproteins gp50, gp63,gI or immunogenic fragments thereof operatively linked to an expressioncontrol sequence.

The present invention also provides vaccines comprising gp50 and gp63and methods of protecting animals from PRV infection by vaccinating themwith these polypeptides.

DETAILED DESCRIPTION OF INVENTION

The existence and location of the gene encoding glycoprotein gp50 of PRVwas demonstrated by M. W. Wathen and L. M. Wathen, supra.

The glycoprotein encoded by the gene was defined as a glycoprotein thatreacted with a particular monoclonal antibody. This glycoprotein did notcorrespond to any of the previously known PRV glycoproteins. Wathen andWathen mapped a mutation resistant to the monoclonal antibody, which,based on precedent in herpes simplex virus (e.g., T. C. Holland et al.,J. Virol., 52, pp.566-74 (1984)), maps the location of the structuralgene for gp50. Wathen and Wathen mapped the gp50 gene to the smallerSalI/BmHI fragment from within the BamHI 7 fragment of PRV. Rea et al,supra, have mapped the PRV glycoprotein gX gene to the same region.

The PRV gp63 and gI genes were isolated by screening PRV DNA librariesconstructed in the bacteriophage expression vector λgt11 (J. G. Timmins,et al., "A method for Efficient Gene Isolation from Phage λgt11Libraries: Use of Antisera to Denatured, Acetone-Precipitated Proteins",Gene, 39, pp. 89-93 (1985); R. A. Young and R. W. Davis, Proc. Natl.Acad. Sci. U.S.A., 80, pp. 1194-98 (1983); R. A. Young and R. W. Davis,Science, 222, pp.778-82 (1983)).

PRV genomic DNA derived from PRV Rice strain originally obtained from D.P. Gustarson at Purdue University was isolated from the cytoplasm ofPRV-infected Vero cells (ATCC CCL 81). The genomic DNA was fragmented bysonication and then cloned into λgt11 to produce a λ/PRV recombinant(λPRV) DNA library.

Antisera for screening the λPRV library were produced by inoculatingmice with proteins isolated from cells infected with PRV (infected cellproteins or ICP's) that had been segregated according to size on SDSgels, and then isolating the antibodies. The λPRV phages to be screenedwere plated on a lawn of E. coli. λgt11 contains a unique cloning sitein the 3' end of the lacZ gene. Foreign DNA's inserted in this uniquesite in the proper orientation and reading frame produce, on expression,polypeptides fused to β-galactosidase. A nitrocellulose filtercontaining an inducer of lacZ transcription to enhance expression of thePRV DNA was laid on top of the lawn. After the fusion polypeptidesexpressed by λPRV's had sufficient time to bind to the nitrocellulosefilters, the filters were removed from the lawns and probed with themouse antisera. Plaques producing antigen that bound the mice antiserawere identified by probing with a labeled antibody for the mouseantisera.

Plaques that gave a positive signal were used to transform an E. colihost (Y1090, available from the ATCC, Rockville, Md. 20852). Thecultures were then incubated overnight to produce the λPRV phage stocks.These phage stocks were used to infect E. coli K95 (D. Freidman, in TheBacteriophage Lambda, pp. 733-38, A. D. Hershey, (1971)). Polypeptidesproduced by the transformed E. coli K95 were purified by preparative gelelectrophoresis. Polypeptides that were overproduced (due to inductionof transcription of the tacZ gene), having molecular weights greaterthan 116,000 daltons, and which were also absent from λgt11 controlcultures were β-galactosidase-PRV fusion proteins. Each individualfusion protein was then injected into a different mouse to produceantisera.

Labeled PRV ICP's were produced by infecting Vero cells growing in amedium containing, for example, ¹⁴ C-glucosamine (T. J. Rea, et el.,supra.). The fusion protein antisera from above were used toimmunoprecipitate these labeled ICP's. The polypeptides so precipitatedwere analyzed by gel electrophoresis. One of them was a 110 kd MWglycoprotein (gI) and another a 63 kd MW glycoprotein (gp63). The genescloned in the phages that produced the hybrid proteins raising anti-gIand anti-gp63 serum were thus shown to be the gI and gp63 genes. Thesegenes were found to map within the BamHI 7 fragment of the PRV genome(T. J. Rea, et el., supra.) as does the gp50 sequence (see Chart D). ThegI location is in general agreement with the area where Mettenleiter, etal., supra, had mapped the gI gene. However, Mettenleiter, et al.implied that the gI gene extends into the BamHI 12 fragment which itdoes not.

This λPRV gene isolation method is rapid and efficient when compared toDNA hybridization and to in vitro translation of selected mRNAs. Becausepurified glycoproteins were unavailable, we could not construct,rapidly, oligonucleotide probes from amino acid sequence data, nor couldwe raise highly specific polyclonal antisera. Therefore we used themethod set forth above.

As mentioned above, the genes encoding gp50, gp63, and gI mapped to theBamHI 7 fragment of the PRV DNA. The BamHI 7 fragment from PRV can bederived from plasmid pPRXh1 (also known as pUC1129) and fragmentsconvenient for DNA sequence analysis can be derived by standardsubcloning procedures. Plasmid pUC1129 is available from E. coli HB101,NRRL B-15772. This culture is available from the permanent collection ofthe Northern Regional Research Center Fermenta Laboratory (NRRL), U.S.Department of Agriculture, in Peoria, Ill., U.S.A.

E, coli HB101 containing pUC1129 can be grown up in L-broth by wellknown procedures. Typically the culture is grown to an optical densityof 0.6 after which chloramphenicol is added and the culture is left toshake overnight. The culture is then lysed by, e.g., using high salt SDSand the supernatant is subjected to a cesium chloride/ethidium bromideequilibrium density gradient centrifugation to yield the plasmids.

The availability of these gene sequences permits direct manipulation ofthe genes and gene sequences which allows modifications of theregulation of expression and/or the structure of the protein encoded bythe gene or a fragment thereof. Knowledge of these gene sequences alsoallows one to clone the corresponding gene, or fragment thereof, fromany strain of PRV using the known sequence as a hybridization probe, andto express the entire protein or fragment thereof by recombinanttechniques generally known in the art.

Knowledge of these gene sequences enabled us to deduce the amino acidsequence of the corresponding polypeptides (Charts A-C). As a result,fragments of these polypeptides having PRV immunogenicity can beproduced by standard methods of protein synthesis or recombinant DNAtechniques. As used herein, immunogenicity and antigenicity are usedinterchangeably to refer to the ability to stimulate any type ofadaptive immune response, i.e., antigert and antigenicity are notlimited in meaning to substances that stimulate the production ofantibodies.

The primary structures (sequences) of the genes coding for gp50, gp63,and gI also are set forth in Charts A-C.

The genes or fragments thereof can be extracted from pUC1129 bydigesting the plasmid DNA from a culture of NRRL B-15772 withappropriate endonuclease restriction enzymes. For example, the BamHI 7fragment may be isolated by digestion of a preparation of pUC1129 withBamHI, and isolation by gel electrophoresis.

All restriction endonucleases referred to herein are commerciallyavailable and their use is well known in the art. Directions for usegenerally are provided by commercial suppliers of the restrictionenzymes.

The excised gene or fragments thereof can be ligated to various cloningvehicles or vectors for use in transforming a host cell. The vectorspreferably contains DNA sequences to initiate, control and terminatetranscription and translation (which together comprise expression) ofthe PRV glycoprotein genes and are, therefore, operatively linkedthereto. These "expression control sequences" are preferably compatiblewith the host cell to be transformed. When the host cell is a higheranimal cell, e.g., a mammalian cell, the naturally occurring expressioncontrol sequences of the glycoprotein genes can be employed alone ortogether with heterologous expression control sequences. Heterologoussequences may also be employed alone. The vectors additionallypreferably contain a marker gene (e.g., antibiotic resistance) toprovide a phenotypic trait for selection of transformed host cells.Additionally a replicating vector will contain a replicon.

Typical vectors are plasmids, phages, and viruses that infect animalcells. In essence, one can use any DNA sequence that is capable oftransforming a host cell.

The term host cell as used herein means a cell capable of beingtransformed with the DNA sequence coding for a polypeptide displayingPRV glycoprotein antigenicity. Preferably, the host cell is capable ofexpressing the PRV polypeptide or fragments thereof. The host cell canbe procaryotic or eucaryotic. Illustrative procaryotic cells arebacteria such as E. coli, B. subtilis, Pseudomonas, and B.stearothermophilus. Illustrative eucaryotic cells are yeast or higheranimal cells such as cells of insect, plant or mammalian origin.Mammalian cell systems often will be in the form of monolayers of cellsalthough mammalian cell suspensions may also be used. Mammalian celllines include, for example, VERO and HeLa cells, Chinese hamster ovary(CHO) cell lines, WI38, BHK, COS-7 or MDCK cell lines. Insect cell linesinclude the Sf9 line of Spodoptera frugiperda (ATCC CRL1711). A summaryof some available eucaryotic plasmids, host cells and methods foremploying them for cloning and expressing PRV glycoproteins can be foundin K. Esser, et al., Plasmids of Eukaryotes (Fundamentals andApplications), Springer-Verlag (1986) which is incorporated herein byreference.

As indicated above, the vector, e.g., a plasmid, which is used totransform the host cell preferably contains compatible expressioncontrol sequences for expression of the PRV glycoprotein gene orfragments thereof. The expression control sequences are, therefore,operatively linked to the gene or fragment. When the host cells arebacteria, illustrative useful expression control sequences include thetrp promoter and operator (Goeddel, et al., Nucl. Acids Res., 8, 4057(1980)); the lac promoter and operator (Chang, et al., Nature, 275, 615(1978)); the outer membrane protein promoter (EMBO J., 1, 771-775(1982)); the bacteriophage λ promoters and operators (Nucl. Acids Res.,11, 4677-4688 (1983)); the α-amylase (B. subtilis) promoter andoperator, termination sequences and other expression enhancement andcontrol sequences compatible with the selected host cell. When the hostcell is yeast, illustrative useful expression control sequences include,e.g., α-mating factor. For insect cells the polyhedrin promoter ofbaculoviruses can be used (Mol. Cell. Biol., 3, pp. 2156-65 (1983)).When the host cell is of insect or mammalian origin illustrative usefulexpression control sequences include, e.g. , the SV-40 promoter(Science, 222, 524-527 (1983)) or, e.g. , the metallothionein promoter(Nature , 296 , 39-42 (1982)) or a heat shock promoter (Voellmy, et al.,Proc. Natl. Acad. Sci. U.S.A., 82, pp. 4949-53 (1985)). As noted above,when the host cell is mammalian one may use the expression controlsequences for the PRV glycoprotein gene but preferably in combinationwith heterologous expression control sequences.

The plasmid or replicating or integrating DNA material containing theexpression control sequences is cleaved using restriction enzymes,adjusted in size as necessary or desirable, and ligated with the PRVglycoprotein gene or fragments thereof by means well known in the art.When yeast or higher animal host cells are employed, polyadenylation orterminator sequences from known yeast or mammalian genes may beincorporated into the vector. For example, the bovine growth hormonepolyadenylation sequence may be used as set forth in Europeanpublication number 0 093 619 and incorporated herein by reference.Additionally gene sequences to control replication of the host cell maybe incorporated into the vector.

The host cells are competent or rendered competent for transformation byvarious means. When bacterial cells are the host cells they can berendered competent by treatment with salts, typically a calcium salt, asgenerally described by Cohen, PNAS, 69, 2110 (1972). A yeast host cellgenerally is rendered competent by removal of its cell wall or by othermeans such as ionic treatment (J. Bacteriol., 153, 163-168 (1983)).There are several well-known methods of introducing DNA into animalcells including, e.g., calcium phosphate precipitation, fusion of therecipient cells with bacterial protoplasts containing the DNA, treatmentof the recipient cells with liposomes containing the DNA, andmicroinjection of the DNA directly into the cells.

The transformed cells are grown up by means well known in the art(Molecular Cloning, Manjarls, T., et al., Cold Spring Harbor Laboratory,(1982); Biochemical Methods In Cell Culture And Virology, Kuchler, R.J., Dowden, Hutchinson and Ross, Inc., (1977); Methods In YeastGenetics, Sherman, F., et al., Cold Spring Harbor Laboratory, (1982))and the expressed PRV glycoprotein or fragment thereof is harvested fromthe cell medium in those systems where the protein is excreted from thehost cell, or from the cell suspension after disruption of the host cellsystem by, e.g., mechanical or enzymatic means which are well known inthe art.

As noted above, the amino acid sequences of the PRV glycoproteins asdeduced from the gene structures are set forth in Charts A-C.Polypeptides displaying PRV glycoprotein antigenicity include thesequences set forth in Chart A-C and any portions of the polypeptidesequences which are capable of eliciting an immune response in ananimal, e.g., a mammal, which has been injected with the polypeptidesequence and also immunogenically functional analogs of thepolypeptides.

As indicated hereinabove the entire gene coding for the PRV glycoproteincan be employed in constructing the vectors and transforming the hostcells to express the PRV glycoprotein, or fragments of the gene codingfor the PRV glycoprotein can be employed, whereby the resulting hostcell will express polypeptides displaying PRV antigenicity. Any fragmentof the PRV glycoprotein gene can be employed which results in theexpression of a polypeptide which is an immunogenic fragment of the PRVglycoprotein or an analog thereof. As is well known in the art, thedegeneracy of the genetic code permits easy substitution of base pairsto produce functionally equivalent genes and fragments thereof encodingpolypeptides displaying PRV glycoprotein antigenicity. These functionalequivalents also are included within the scope of the invention.

Charts D-S are set forth to illustrate the constructions of theExamples. Certain conventions are used to illustrate plasmids and DNAfragments as follows:

(1) The single line figures represent both circular and lineardouble-stranded DNA.

(2) Asterisks (*) indicate that the molecule represented is circular.Lack of an asterisk indicates the molecule is linear.

(3) Endonuclease restriction sites of interest are indicated above theline.

(4) Genes are indicated below the line.

(5) Distances between genes and restriction sites are not to scale. Thefigures show the relative positions only unless indicated otherwise.

Most of the recombinant DNA methods employed in practicing the presentinvention are standard procedures, well known to those skilled in theart, and described in detail, for example, in Molecular Cloning, T.Maniatis, et el., Cold Spring Harbor Laboratory, (1982) and B. Perhal, APractical Guide to Molecular Cloning, John Wiley & Sons (1984), whichare incorporated herein by reference.

EXAMPLE 1

In this example we set forth the sequencing, cloning and expression ofPRV glycoprotein gp50.

1. Sequencing of the gp50 Gene

The BamHI 7 fragment of PRV Rice strain DNA (Chart D) which encodes thegp50 gene is isolated from pPRXh1 [NRRL B- 15772 ], supra., andsubcloned into the BamHI site of plasmid pBR322 (Maniatis et el.,supra.).

Referring now to Chart E, the fragment is further subcloned usingstandard procedures by digesting BamHI 7 with PvuII, isolating the twoBamHI/PvulI fragments (1.5 and 4.9 kb) and subcloning them between theBamHI and PvuII sites of pBR322 to produce plasmids pPR28-4 and pPR28-1incorporating the 1.5 and 4.9 kb fragments respectively (see also, Reaet el., supra.). These subclones are used as sources of DNA for DNAsequencing experiments.

Chart F shows various restriction enzyme cleavage sites located in thegp50 gene and flanking regions. The 1.5 and 4.9 kb fragments subclonedabove are digested with these restriction enzymes. Each of the endsgenerated by the restriction enzymes is labeled with γ-³² P-ATP usingpolynucleotide kinase and sequenced according to the method of Maxam andGilbert, Methods Enzymol., 65, 499-560 (1980). The entire gene issequenced at least twice on both strands. The DNA sequence for gp50 isset forth in Chart A. This DNA may be employed to detect animalsactively infected with PRV. For example, one could take a nasal orthroat swab, and then do a DNA/DNA hybridization by standard methods todetect the presence of PRV.

2. Expression of gp50

Referring now to Chart G, a NarI cleavage site is located 35 base pairsupstream from the gp50 gene initiation codon. The first step inexpression is insertion of the convenient BamHI cleavage site at thepoint of the NarI cleavage site. Plasmid pPR28-4 from above is digestedwith restriction endonuclease NarI to produce DNA fragment 3 comprisingthe N-terminus encoding end of the gp50 gene and a portion of the gXgene. BamHI linkers are added to fragment 3 and the fragment is digestedwith BamHI to delete the gX sequence thus producing fragment 4. TheBamHI ends are then ligated to produce plasmid pPR28-4 Nar2.

Referring now to Chart H, we show the assembly of the complete gp50gene. pPR28-4 Nar2 is digested with BamHI and PvuII to produce fragment5 (160 bp) comprising the N-terminal encoding portion of the gp50 gene.Plasmid pPR28-1 from above is also digested with PvuII and BamHI toproduce a 4.9 kb fragment comprising the C-terminal encoding portion ofthe gp50 gene (fragment 6). Plasmid pPGX1 (constructed as set forth inU.S. patent application Ser. No. 760,130)), or, alternatively, plasmidpBR322, is digested with BamHI, treated with bacterial alkalinephosphatase (BAP) and then ligated with fragments 5 and 6 to produceplasmid pBGP50-23 comprising the complete gp50 gene.

Referring now to Chart I, we show the production of plasmid pD50.Plasmid pBG50-23 is cut with restriction enzyme MaeIII (K. Schmid etal., Nucl. Acids Res., 12, p. 2619 (1984)) to yield a mixture offragments. The MaeIII ends are made blunt with T4 DNA polymerase andEcoRI linkers are added to the blunt ends followed by EcoRI digestion.The resulting fragments are cut with BamHI and a 1.3 kb BamHI/EcoRIfragment containing the gp50 gene (fragment 7) is isolated. PlasmidpSV2dhfr (obtained from the American Type Culture Collection, BethesdaResearch Laboratories, or synthesized according to the method of S.Subramani, et al., Mol. Cell. Biol., 2, pp 854-64 (1981)) is digestedwith BamHI and EcoRI and the larger (5.0 kb) fragment is isolated toproduce fragment 8 containing the dihydrofolate reductase (dhfr) marker.Fragments 7 and 8 are then ligated to produce plasmid pD50 comprisingthe gp50 gene and the dhfr marker.

Referring now to Chart J, the immediate early promoter from humancytomegalovirus Towne strain is added upstream from the gp50 gene. pD50is digested with BamHI and treated with bacterial alkaline phosphataseto produce fragment 9. A 760 bp Sau3A fragment containing the humancytomegalovirus (Towne) immediate early promoter is isolated accordingto the procedure set forth in U.S. patent application Ser. No. 758,517to produce fragment 10 (see also, D. R. Thomsen, et al., Proc. Natl.Acad. Sci. U.S.A., 81, pp. 659-63 (1984)). These fragments are thenligated by a BamHI/Sau3A fusion to produce plasmid pDIE50. To confirmthat the promoter is in the proper orientation to transcribe the gp50gene the plasmid is digested with SacI and PvuII and a 185 bp fragmentis produced.

Referring now to Chart K, the 0.6 kb PvuII/EcoRI fragment containing thebovine growth hormone polyadenylation signal is isolated from plasmidpGH2R2 (R. P. Woychik, et al., Nucl. Acids Res., 10, pp. 7197-7210(1982) by digestion with PvuII and EcoRI or from pSVCOW7 (supra.) toproduce fragment 11.

Fragment 11 is cloned between the EcoRI and SmaI cleavage sites of pUC9(obtained from Pharmacia/PL or ATCC) t o give pCOWT1. pCOWT1 is cut withSalI, the ends made blunt with T4 DNA polymerase, EcoRI linkers areadded, the DNA is cut with EcoRI, and the 0.6 kb fragment (fragment 12)is isolated. This is the same as fragment 11 except that it has twoEcoRI ends and a polylinker sequence at one end.

Plasmid pDIE50 is cut with EcoRI, and fragment 12 is cloned into it toproduce plasmid pDIE50PA. Digestion with BamHI and PvuII produces afragment of 1.1 kb in the case where the polyadenylation signal is inthe proper orientation. The plasmid can also be constructed by cloningin the polyadenylation sequence before the promoter.

Plasmid pDIE50PA is used to transfect CHO dhfr⁻ cells (DXB-11, G. Urlauband L. A. Chasin, Proc. Natl. Acad. Sci. U.S.A., 77, pp. 4216-20 (1980))by calcium phosphate co-precipitation with salmon sperm carrier DNA (F.L. Graham and A. J. Van Der Eb, Virol., 52, pp. 456-67 (1973)). Thedihydrofolate reductase positive (dhfr⁺) transfected cells are selectedin Dulbecco's modified Eagle's medium plus Eagle's non-essential aminoacids plus 10% fetal calf serum. Selected dhfr⁺ CHO cells produce gp50as detected by immunofluorescence with anti-gp50 monoclonal antibody3A-4, or by labelling with ¹⁴ C-glucosamine and immunoprecipitation with3A-4. Monoclonal antibody 3A-4 is produced as described in copendingU.S. patent application Ser. No. 817,429, filed Jan. 9, 1985.Immunoprecipitation reactions are performed as described previously (T.J. Rea, et al., supra.) except for the following The extracts are firstincubated with normal mouse serum, followed by washed Staphylococcusaureus cells, and centrifuged for 30 minutes in a Beckman SW50.1 rotorat 40,000 rpm. After extracts are incubated with monoclonal orpolyclonal antiserum plus S. Aureus cells, the cells are washed threetimes in 10 mM Tris HCl, pH 7.0, 1 mM EDTA, 0.1M NaCl, 1% NP40 and 0.5%deoxycholate. Analysis of proteins is done on 11% SDS polyacrylamidegels (L. Morse, et al., J. Virol., 26, pp. 389-410 (1984)). Inpreliminary immunofluorescence assays it was found that 3A-4 reactedwith the pDIE50PA-transfected CHO cells but not with untransfected CHOcells. When the transfected CHO cells were labelled with ¹⁴C-glucosamine, 3A-4 immunoprecipitated a labelled protein from cellscontaining pDIE50PA but not from control cells making human renin. Theprecipitated protein co-migrated on SDS-polyacrylamide gels with theprotein precipitated by 3A-4 from PRV-infected cells.

A clone of these transfected CHO cells producing gp50 can be grown inroller bottles, harvested in phosphate buffered saline plus 1 mM EDTA,and mixed with complete Freund's adjuvant for use as a vaccine.

The gp50 gene can also be expressed in a vaccinia vector. In thisembodiment, after pBG50-23 is digested with MaeIII and the ends madeblunt with T4 DNA polymerase, the DNA is digested with BamHI. The 1.3BamHI/blunt-ended fragment containing the gp50 gene is isolated. PlasmidpGS20 (Mackett, et al., J. Virol., 49, pp. 857-64 (1984)) is cut withBamHI and SmaI, and the larger 6.5 kb fragment is isolated by gelelectrophoresis. These two fragments are ligated together to producepVV50. Plasmid pVV50 is transfected into CV-1 cells (ATCC CCL 70)infected with the WR strain of vaccinia virus (ATCC VR-119), andselected for thymidine kinase negative recombinants by plating on 143cells (ATCC CRL 8303) in 5-bromodeoxyuridine (BUdR) by the methodsdescribed by Mackett, et al . in DNA Cloning, Volume II: A PracticalApproach, D. M. Glover, ed. , IRL Press, Oxford (1985). The resultingvirus, vaccinia-gp50, expressed gp50 in infected cells, as assayed bylabelling of the proteins of the infected cell with ¹⁴ C-glucosamine andimmunoprecipitation with monoclonal antibody 3A-4.

EXAMPLE 2

In this example we set forth the protection of mice and swine from PRVchallenge using the gp50 of Example i as an immunogenic agent.

In Tables 1-3, infra, the microneutralization assay was done as follows:Serial two-fold dilutions of serum samples were done in microtiterplates (Costar) using basal medium Eagle (BME) supplemented with 3%fetal calf serum and antibiotics. About 1000 pfu (50 μl) of PRV wereadded to 50 μl of each dilution. Rabbit complement was included in thevirus aliquot at a dilution of 1:5 for the mouse serum assays but notthe pig serum assays. The samples were incubated for either 1 hr (swinesera) or 3 hrs (mouse sera) at 37° C. After the incubation period, analiquot (50 μl) of porcine kidney-15 (PK-15) cells (300,000 cells/ml) inEagle's Minimum Essential Medium was added to each serum per PRV sample.The samples were subsequently incubated at 37° C. for 2 days.Neutralizing titers represent the reciprocals of the highest dilutionswhich protected 50% of the cells from cytopathic effects.

Table 1 sets forth the protection of mice from challenge by virulent PRVby immunization with gp50 produced in vaccinia virus. Mice wereimmunized by tail scarification with 25 μl or by the footpad route with50 μl. Mice were immunized 28 days prior to challenge (except mice givenPR-Vac which were immunized 14 days prior to challenge).

                  TABLE 1                                                         ______________________________________                                        Immunizing                                                                             Dose               Neutralizing                                                                           %                                        Agent    (PFU)    Route     Titers.sup.a                                                                           Survival.sup.b                           ______________________________________                                        gp50     3.0 × 10.sup.7                                                                   Tail      1024     93                                       gp50     6.0 × 10.sup.7                                                                   Footpad   1024     100                                      gp50     7.5 × 10.sup.6                                                                   Tail       512     93                                       vaccinia.sup.c                                                                         7.5 × 10.sup.6                                                                   Tail       <8      27                                       BME.sup.d                                                                              --       Tail       <8      20                                       PR-Vac.sup.e                                                                           --       Footpad    512     90                                       ______________________________________                                         .sup.a Neutralizing titer against PRV at day of challenge (+ complement).     .sup.b Challenged with 10 LD50 of PRV Rice strain by intraperitoneal          route.                                                                        .sup.c Control virus.                                                         .sup.d Basal medium Eagle, negative control.                                  .sup.e Norden Laboratories, Lincoln, NE, inactivated PRV vaccine, positiv     control.                                                                 

Table 2 sets forth the protection of mice from challenge by virulent PRVby immunization with gp50 produced in CHO cells. Mice were immunized at281 days, 18 days and 7 days prior to challenge. Mice receivedpreparation with adjuvants subcutaneously on the first dose andpreparations in saline intrapertioneally on the second and third doses.Each mouse received 10⁶ disrupted cells/dose.

                  TABLE 2                                                         ______________________________________                                        Immunizing    Neutralizing  %                                                 Agent/Adjuvant                                                                              Titers.sup.a  Survival.sup.b                                    ______________________________________                                        gp50/CFA.sup.c                                                                               512          100    (10/10)                                    gp50/CFA (2 doses)                                                                          ND            80     (4/5)                                      gp50/IFA.sup.d                                                                              1024          90     (9/10)                                     gp50/saline    256          100    (3/3)                                      CHO-renin.sup.e /CFA                                                                         <8           10     (1/10)                                     Nontreated     <8           0      (0/10)                                     PR-Vac.sup.f  4096          90     (9/10)                                     ______________________________________                                         .sup.a Neutralizing titer against PRV at day of challenge (+ complement).     .sup.b Challenged with 30 LD50 of PRV Rice strain by footpad route.           .sup.c Complete Freund's adjuvant.                                            .sup.d Incomplete Freund's adjuvant.                                          .sup.e Control cells expressing renin.                                        .sup.f Norden Laboratories, Lincoln, NE, inactivated PRV vaccine, positiv     control.                                                                 

Table 3 sets forth the protection of swine from challenge by virulentPRV by immunization with gp50 produced in CHO cells. Swine wereimmunized at 21 days and 7 days prior to challenge. Swine received 2×10⁷disrupted cells per dose. The first dose was mixed with completeFreund's adjuvant while the second dose was suspended in saline. Bothdoses were given intramuscularly.

                  TABLE 3                                                         ______________________________________                                        Immunizing     Geometric Mean                                                                             %                                                 Agent/Adjuvant Titer.sup.a  Survival.sup.b                                    ______________________________________                                        gp50/CFA        25          100                                               CHO-renin/CFA  <8            0                                                ______________________________________                                         .sup.a Neutralizing titer against PRV at day of challenge.                    .sup.b Challenge with PRV Rice strain 1 × 10.sup.5 pfu/pig by the       intranasal route.                                                        

These three tables demonstrate that gp50 can raise neutralizingantibodies and protect mice and swine from lethal PRV challenge.

In another aspect of the instant invention we produced a derivative ofglycoprotein gp50 by removing the DNA coding for the C-terminal end ofgp50. The resulting polypeptide has a deletion for the amino acidsequence necessary to anchor gp50 into the cell membrane. When expressedin mammalian cells this gp50 derivative is secreted into the medium.Purification of this gp50 derivative from the medium for use as asubunit vaccine is much simpler than fractionation of whole cells.Removal of the anchor sequence to convert a membrane protein into asecreted protein was first demonstrated for the influenza hemagglutiningene (M. J. Gething and J. Sambrook, Nature, 300, pp. 598-603 (1982)).

Referring now to Chart L, plasmid pDIE50 from above is digested withSalI and EcoRI. The 5.0 and 0.7 kb fragments are isolated. The 0.7 kbfragment encoding a portion of gp50 is digested with Sau3A and a 0.5 kbSalI/Sau3A fragment is isolated. To introduce a stop codon after thetruncated gp50 gene, the following oligonucleotides are synthesized:##STR1##

The 5.0 kb EcoRI/SalI fragment, the 0.5 kb SalI/Sau3A fragment and theannealed oligonucleotides are ligated to produce plasmid pDIE5OT.Digestion with EcoRI and SalI produces a 580 bp fragment. pDIE50T is cutwith EcoRI and the 0.6 kb EcoRI fragment containing the bGH polyA site(fragment 12) is cloned in to produce plasmid pDIE50TPA. Digestion ofpDIE50TPA with BamHI and PvuII yields a 970 bp fragment when thepolyadenylation signal is in the proper orientation.

pDIE50TPA is used to transfect CHO dhfr⁻ cells. Selected dhfr⁺ CHO cellsproduce a truncated form of gp50 which is secreted into the medium asdetected by labelling with immunoprecipitation. ³⁵ S-methionine andimmunoprecipitation.

EXAMPLE 3

In this example we set forth the isolation, cloning and sequencing ofthe gp63 and gI genes.

1. Library Construction

PRV genomic DNA was prepared as described previously (T. J. Rea, et al.,supra.). Fragments of 0.5-3.0 kb were obtained by sonicating the PRVgenomic DNA of the PRV Rice strain twice for 4 sec each time at setting2 with a Branson 200 sonicator. After blunt ending the fragments with T4DNA polymerase, the fragments were ligated to kinased EcoRI linkers (T.Manjarls, et al., supra). After over-digestion with EcoRI (since PRV DNAdoes not contain an EcoRI site, methylation was unnecessary), excesslinkers were removed by agarose gel electrophoresis. The PRV DNAfragments in the desired size range were eluted by the glass slurrymethod, (B. Vogelstein and D. Gillespie, Proc. Natl. Acad. Sci. U.S.A.,76, pp. 615-19 (1979)). A library of 61,000 λ/PRV recombinants (αPRVs)was constructed by ligating 500 ng of PRV DNA fragments to 750 ng ofEcoRI digested λgt11 (R. A. Young and R. W. Davis, supra.) DNA in 50 mMTris (pH 7.4), 10 mM MgCl₂ , 10 mM dithiothreitol, 1 mM spermidine, 1 mMATP, 400 units of T4 DNA ligase (New England Biolabs), in a final volumeof 10 μl. The ligated DNA was packaged into bacteriophage λ virionsusing the Packagene extract (Promega Biotec, Madison, Wisc.).

2. λPRV Library Screening

The λPRV library was screened as previously described (J. G. Timmins, etal., supra.; R. A. Young and R. W. Davis, supra.). 20,000 phages werescreened per 150 mm LB-ampicillin plate. The screening antisera wereraised by injecting mice with size fractions of PRV infected cellproteins (ICP's) eluted from SDS-polyacrylamide gels (J. G. Timmins, etal., supra.). Plaques giving positive signals upon screening withantisera were picked from the agar plates with a sterile pasteurpipette, resuspended in 1 ml SM buffer (T. Maniatis, et al., supra) andrescreened. The screening was repeated until the plaques werehomogeneous in reacting positively.

Approximately 43,000 λPRV recombinants were screened with mouse antiserato PRV infected Vero cell proteins, isolated from SDS-polyacrylamidegels. Sixty positive λPRV phages were isolated.

3. Phage Stock Preparation

High titer phage stocks (10¹⁰ -10¹¹ pfu/ml) were prepared by the platelysate method (T. Maniatis et al., supra). A single, well-isolatedpositive signal plaque was picked and resuspended in 1 ml SM. 100 μl ofthe suspension was adsorbed to 300 μl of E. coli Y1090 (available fromthe American Type Culture Collection (ATCC), Rockville, Md.) at 37° C.for 15 min, diluted with 10 ml LB-top agarose, poured evenly on a 150 mmLB-ampicillin plate and incubated overnight at 42° C. The top agarosewas gently scraped off with a flamed glass slide and transferred to a 30ml Corex tube. 8 ml of SM and 250 μl of chloroform were added, mixed andincubated at 37° C. for 15 min. The lysate was clarified bycentrifugation at 10,000 rpm for 30 min in the HB-4 rotor. The phagestock was stored at 4° C. with 0.3% chloroform.

4. Fusion Protein Preparation and Analysis

LB medium (Maniatis, et al., supra.) was inoculated 1:50 with a freshovernight culture of E. coli K95 (sup⁻, λ⁻, gal⁻, str^(r), nusA⁻ ; D.Friedman, supra.) and grown to an OD₅₅₀ =0.5 at 30° C. 25 ml of culturewas infected with λPRV phage at a multiplicity of 5 and incubated in a42° C. shaking water bath for 25 min, followed by transfer to 37° C. for2-3 hours. The cells were pelleted at 5,000 rpm for 10 min in the HB-4rotor and resuspended in 100 μl of 100 mM Tris (pH 7.8), 300 mM NaCl. Anequal volume of 2x SDS-PAGE sample buffer was added, and the sample wasboiled for 10 min. 5 μl of each sample was analyzed by electrophoresison analytical SDS-polyacrylamide gels as described in L. Morse et al.,J. Virol, 26, pp. 389-410 (1978). The fusion polypeptide preparationswere scaled up 10-fold for mouse injections. The β-galactosidase/PRVfusion polypeptides were isolated after staining a strip of the gel withcoomassie blue (L. Morse et al., supra; K. Weber and M. Osborn, in TheProteins, 1, pp. 179-223 (1975)). Fusion polypeptide quantities wereestimated by analytical SDS-PAGE. Cell lysates from λPRV infected E.coli K95 cultures were electrophoresed in 9.25% SDS-polyacrylamide gels.Overproduced polypeptide bands with molecular weights greater than116,000 daltons, absent from λgt11-infected controls, wereβ-galactosidase-PRV fusion polypeptides. The β-galactosidase-PRV fusionpolypeptides ranged in size from 129,000 to 158,000 daltons.Approximately 50-75 μg of fusion polypeptide was resuspended in completeFreund's adjuvant and injected subcutaneously and interperitoneally permouse. Later injections were done intraperitoneally in incompleteFreund's adjuvant.

5. Antisera Analysis

Immunoprecipitations of ¹⁴ C-glucosamine ICP's, ³⁵ S-methionine ICP'sand ¹⁴ C-glucosamine gX were done as previously described (T. J. Rea, etal., supra.). These techniques showed that gp63 and gI had been isolatedin a λgt11 recombinant phage. We called these phages λ37 and λ36 (gp63)and λ23 (gI).

6. λDNA Mini-preps

Bacteriophage were rapidly isolated from plate lysates (T. J. Silhavy etal., Experiments With Gene Fusions, (1984)). 5% and 40% glycerol steps(3 ml each in SM buffer) were layered in an SW41 tube. A plate lysate(˜6 ml) was layered and centrifuged at 35,000 rpm for 60 min at 4° C.The supernatant was discarded and the phage pellet was resuspended in 1ml SM. DNAse I and RNAse A were added to final concentrations of 1 μg/mland 10 μg/ml. After incubation at 37° C. for 30 min, 200 μl of SDS Mix(0.25M EDTA, 0.5M Tris (pH 7.8), 2.5% SDS) and proteinase K (to 1 mg/ml)were added and incubated at 68° C. for 30 min. The λDNA was extractedwith phenol three times, extracted with chloroform, and ethanolprecipitated. An average 150 mm plate lysate yields 5-10 μ g of λDNA.

7. λPRV DNA Analysis

PRV DNA was digested to completion with BamHI and KpnI, electrophoresedin 0.8% agarose and transferred to nitrocellulose by the method ofSouthern (J. Mol. Biol., 98, pp. 503-17 (1975)). The blots were slicedinto 4 mm strips and stored desiccated at 20°-25° . λPRV DNAs werenick-translated (Amersham) to specific activities of approximately 10⁸cpm/μg. Pre-hybridization was done in 6x SSC, 30% formamide, 1xDenhardt's reagent (0.02% each of ficoll, polyvinylpyrrolidone andbovine serum albumin), 0.1% SDS, 50 μg/ml heterologous DNA at 70° C. for1 hour. Hybridization was done in the same solution at 70° C. for 16hours. Fifteen minute washes were done twice in 2x SSC, 0.1% SDS andtwice in 0.1x SSC, and 0.1% SDS, all at 20°-25°. The blots wereautoradiographed with an intensifying screen at ˜70° C. overnight.

By Southern blotting the PRV glycoprotein genes contained in λ23, λ36and λ37 mapped to the BamHI 7 fragment in the unique small region (seeT. J. Rea, et al., supra.). Finer mapping of this fragment showed thatλ23 (gI) gene mapped distal to the gX gene and that λ37 mapped to theinternal region of BamHI 7, as shown in Chart D.

8. Sequencing The gp63 and gI Genes

The PRV DNA in λ36 and λ37 was determined to contain a StuI cleavagesite. There is only one StuI cleavage site in the BamHI 7 fragment;therefore, the open reading frame that included the StuI cleavage sitewas sequenced. Chart E shows various restriction enzyme cleavage siteslocated in the gp63 gene and flanking regions. BamHI 7 was subcloned anddigested with these restriction enzymes. Each of the ends generated bythe restriction enzymes was labeled with γ-³² P-ATP using polynucleotidektnase and sequenced according to the method of Maxam and Gilbert,Methods Enzymol., 65, 499-560 (1980).

Plasmid pPR28 is produced by cloning the BamHI 7 fragment isolated frompUC1129 into plasmid pSV2 gpt (R. C. Mulligan and P. Berg, Proc. Natl.Acad. Sct. U.S.A., 78, pp. 2072-76 (1981)).

Plasmid pPR28-1 was produced by digesting pPR28 with PvuII and thenrecircularizing the piece containing the E. coli origin of replicationand bla gene to produce a plasmid comprising a 4.9 kb PvuII/BamHI 7 PRVfragment containing the DNA sequence for gI.

Chart N shows various restriction enzyme cleavage sites located in thegI gene and flanking regions. BamHI 7 was subcloned, digested, labeledand sequenced as set forth above.

The DNA sequences for glycoproteins gp63 and gI are set forth in ChartsB and C respectively. This DNA may be employed to detect animalsactively infected with PRV. For example, one could take a nasal orthroat swab, and then by standard DNA/DNA hybridization methods detectthe presence of PRV.

EXAMPLE 4

In this example we set forth the expression of gI in mammalian cells.

A BamHI 7 fragment containing the gI gene is isolated from plasmid pPR28(see above) by digesting the plasmid with BamHI, separating thefragments on agarose gel and then excising the fragment from the gel.

Referring now to Chart 0, the BamHI 7 fragment isolated above is thencloned into plasmid pUC19 (purchased from Pharmacia/PL) to produceplasmid A. Plasmid A is digested with DraI. DraI cleaves the pUC19sequence in several places, but only once in the BamHI 7 sequencebetween the gp63 and gI genes (Chart D) to produce, inter alia,fragment 1. BamHI linkers are ligated onto the DraI ends of thefragments, including fragment 1, and the resulting fragment mixture isdigested with BamHI. The product fragments are separated by agarose gelelectrophoresis and fragment 2 (2.5 kb) containing the gI gene ispurified. Fragment 2 is cloned into pUC19 digested with BamHI to produceplasmid pUCD/B. Of the two plasmids so produced, the plasmid containingthe gI gene in the proper orientation is determined by digesting theplasmids with BsmI and EcoRI; the plasmid in the proper orientationcontains a characteristic 750 bp BsmI/EcoRI fragment.

Referring now to Chart P, plasmid pUCD/B (Chart 0) is digested with BsmIand EcoRI and the larger fragment (fragment 3, 4.4 kb) is purified byagarose gel electrophoresis. The following two oligonucleotides aresynthesized chemically by well-known techniques or are purchased from acommercial custom synthesis service: ##STR2## These oligonucleotides areligated to fragment 3 to replace the coding sequence for the C-terminusof the gI gene which was deleted by the BsmI cleavage. The resultingplasmid, pGI, contains a complete coding region of the gI gene with aBamHI cleavage site upstream and an EcoRI cleavage site downstream fromthe gI coding sequences.

Plasmid pGI is digested with EcoRI and BamHI and a 1.8 kb fragmentcomprising the gI gene (fragment 4) is purified on an agarose gel.

Plasmid pSV2dhfr, (supra.) is cut with EcoRI, and is then cut with BamHIto produce fragment 5 (5.0 kb) containing the dhfr marker, which isisolated by agarose gel electrophoresis. Then fragments 4, and 5 areligated to produce plasmid pDGI which comprises the dihydrofolatereductase and ampicillin resistance markers, the SV40 promoter andorigin of replication, and the gI gene.

Referring now to Chart Q, the immediate early promoter from humancytomegalovirus Towne strain is added upstream from the gI gene. PlasmidpDGI is digested with BamHI to produce fragment 6. The humancytomegalovirus (Towne) immediate early promoter is isolated (supra.) toproduce fragment 7. Fragments 6 and 7 are then ligated to produceplasmid pDIEGIdhfr. To confirm that the promoter is in the properorientation the plasmid is digested with SacI and BstEII restrictionenzymes. The production of an about 400 bp fragment indicates properorientation.

A 0.6 kb PvuII/EcoRI fragment containing the bovine growth hormonepolyadenylation signal is isolated from the plasmid pSVCOW7 (supra.) toproduce fragment 8. Fragment 8 is cloned across the SmaI/EcoRI sites ofpUG9 (supra.) to produce plasmid pCOWT1. pCOWT1 is cut with SalI,treated with T4 DNA polymerase, and EcoRI linkers are ligated on. Thefragment mixture so produced is then digested with EcoRI and a 0.6 kbfragment is isolated (fragment 9). Fragment 9 is cloned into the EcoRIsite of pUC19 to produce plasmid pCOWT1E. pCOWT1E is digested with EcoRIto produce fragment 10 (600 bp).

Plasmid pDIEGIdhfr is digested with EcoRI and ligated with fragment 10containing the bGH polyadenylation signal to produce plasmid pDIEGIPA.The plasmid having the gI gene in the proper orientation is demonstratedby the production of a 1400 bp fragment upon digestion with BamHI andBstEII.

The resulting plasmid is transfected into dhfr⁻ Chinese hamster ovarycells and dhfr⁺ cells are selected to obtain cell lines expressing gI(Subramani, et al, Mol. Cell Biol., 1. pp.854-64 (1981)). The expressionof gI is amplified by selecting clones of transfected cells that survivegrowth in progressively higher concentrations of methotrexate(McCormick, et al, Mol. Cell Biol., 4, pp. 166-72 (1984).

EXAMPLE 5

In this example we set forth the expression of gp63 in mammalian cells.

The BamHI 7 fragment of PRV DNA (supra.) is isolated from pPRXh1 [NRRLB-15772], and subcloned into the BamHI site of plasmid pBR322 as inExample 1 for use in sequencing and producing more copies of the gp63gene.

Referring now to Chart R, from within BamHI 7 a 1.9 kb BstEII/KphIfragment (fragment 1) is subcloned by cutting BamHi 7 with BstEII,treating the ends with T4 DNA polymerase, and then cutting with KpnI.Fragment 1 is isolated and cloned between the KpnI and SmaI sites inpUC19 (purchased from Pharmacia/PL, Piscataway, N.J.) to yield plasmidpFR28-1BK.

Plasmid pPR28-1BK is cut with DraI plus MaeIII to yield fragment 2 (1.1kb). The DraI cleavage site is outside the coding region of the gp63gene and downstream from its polyadenylation signal. The MaeIII cleavagesite cuts 21 bases downstream from the ATG initiation codon of the gp63gene. To replace the coding region removed from the gp63 gene, thefollowing two oltgonucleotides are synthesized chemically or purchasedfrom commercial custom synthesis services (fragment 4): ##STR3##

Plasmid pSV2dhfr, supra., is cut with EcoRI, treated with T4 DNApolymerase, then cut with BamHI and the larger (5.0 kb) fragment isisolated to produce fragment 4 containing the dhfr marker. Thenfragments 2, 3, and 4 are ligated to produce plasmid pGP63dhfr.

Referring now to Chart S, the immediate early promoter from humancytomegalovirus Towne strain is added upstream from the gp63 gene.pGP63dhfr is digested with BamHI and treated with bacterial alkalinephosphatase to produce fragment 5. A 760 bp Sau3A fragment containinghuman cytomegalovirus (Towne) immediate early promoter is isolated toproduce fragment 6. These fragments are then ligated to produce plasmidpIEGP63dhfr. To confirm that the promoter is in the proper orientationthe plasmid is digested with SacI and PvuII and a 150 bp fragment isproduced.

The resulting plasmid is transfected into dhfr⁻ Chinese hamster ovarycells and dhfr⁺ cells are selected to obtain cell lines expressing gp63.Since the levels of synthesis of gp63 by this system were too low todetect by the methods we used, we produced the polypeptide in vacciniavirus as set forth below.

EXAMPLE 6

In this example we set forth the expression of gp63 in vaccinia virus.The method used herein incorporates aspects of other syntheses referredto above.

Fragments 1, 2, 3, and 4 are produced according to Example 5.

Plasmid pGS20 (Mackerr, et al., J. Virol., 49, pp. 857-64 (1984)) is cutwith BamHI and SmaI, and the larger 6.5 kb fragment is isolated by gelelectrophoresis. Fragment 2, the oligonucleotides, and the pGS20fragment are ligated together to produce plasmid pW63. This plasmid istransfected into CV-1 cells (ATCC CCL 70) infected with the WR strain ofvaccinia virus (ATCC VR-119), selected for thymidine kinase negativerecombinants by plating on 143 cells (ATCC CRL 8303) in BUdR by themethods described by Mackerr, et al. in DNA Cloning, Volume II: APractical Approach, D. M. Glover, ed., IRL Press, Oxford (1985). Theresulting virus, vaccinia-gp63, expresses gp50 in infected cells, asassayed by labelling of the proteins of the infected cell with ¹⁴C-glucosamine and immunoprecipitation with anti-gp63 antiserum.

The BamHI/EcoRI fragment from plasmid pGI, the DraI/MaeIII fragment fromplasmid pPR28-1BK, or the BamHI/MaeIII fragment from pBGP50-23 alldescribed above, may also be treated with Ba131 and inserted in pTRZ4(produced as set forth in copending U.S. patent application Ser. No.606,307) as described in Rea, et al., supra., and used to transform E.coli. By this method, gp50, gp63, and gI can be produced as a fusionprotein in E. coli.

Also, by substituting, for example, pSV2neo (available from the AmericanType Culture Collection) for pSV2dhfr in the above example, therecombinant plasmid comprising the PRV glycoprotein gene could betransformed into other host cells. Transformed cells would be selectedby resistance to antibiotic G418 which is encoded by the plasmid.

One can also express the polypeptides of the instant invention in insectcells as follows: By putting a BamHI linker on the EcoRI site of pD50and digestion with BamHI, or putting a BamHI linker on the EcoRI site ofpGP63dhfr and digestion with BamHI, or by digestion of pUCD/B withBamHI, one obtains BamHI fragments containing the gp50, gp63, or gIgenes respectively. These BamHI fragments can be cloned into a BamHIsite downstream from a polyhedrin promoter in pAC373 (Mol. Cell. Biol.,5, pp. 2860-65 (1985)). The plasmids so produced can be co-transfectedwith DNA from baculovirus Autographa californtca into Sf9 cells, andrecombinant viruses isolated by methods set forth in the article. Theserecombinant viruses produce gp50, gp63, or gI upon infecting Sf9 cells.

A vaccine prepared utilizing a glycoprotein of the instant invention oran immunogenic fragment thereof can consist of fixed host cells, a hostcell extract, or a partially or completely purified PRV glycoproteinpreparation from the host cells or produced by chemical synthesis. ThePRV glycoprotein immunogen prepared in accordance with the presentinvention is preferably free of PRV virus. Thus, the vaccine immunogenof the invention is composed substantially entirely of the desiredimmunogenic PRV polypeptide and/or other PRV polypeptides displaying PRVantigenicity.

The immunogen can be prepared in vaccine dose form by well-knownprocedures. The vaccine can be administered intramuscularly,subcutaneously or intranasally. For parenteral administration, such asintramuscular injection, the immunogen may be combined with a suitablecarrier, for example, it may be administered in water, saline orbuffered vehicles with or without various adjuvants or immunomodulatingagents including aluminum hydroxide, aluminum phosphate, aluminumpotassium sulfate (alum), beryllium sulfate, silica, kaolin, carbon,water-in-oil emulsions, oil-in-water emulsions, muramyl dipeptide,bacterial endotoxin, lipid X, Corynebacterium parrum (Propionobacteriumacnes), Borderella pertussis, polyribonucleotides, sodium alginate,lanolin, lysolecithin, vitamin A, saponin, liposomes, levamisole,DEAE-dextran, blocked copolymers or other synthetic adjuvants. Suchadjuvants are available commercially from various sources, for example,Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.). Anothersuitable adjuvant is Freund's Incomplete Adjuvant (Difco Laboratories,Detroit, Mich.).

The proportion of immunogen and adjuvant can be varied over a broadrange so long as both are present in effective amounts. For example,aluminum hydroxide can be present in an amount of about 0.5% of thevaccine mixture (Al₂ O₃ basis). On a per dose basis, the concentrationof the immunogen can range from about 1.0 μg to about 100 mg per pig. Apreferable range is from about 100 μg to about 3.0 mg per pig. Asuitable dose size is about 1-10 ml, preferably about 1.0 ml.Accordingly, a dose for intramuscular injection, for example, wouldcomprise 1 ml containing 1.0 mg of immunogen in admixture with 0.5%aluminum hydroxide. Comparable dose forms can also be prepared forparenteral administration to baby pigs, but the amount of immunogen perdose will be smaller, for example, about 0.25 to about 1.0 mg per dose.

For vaccination of sows, a two dose regimen can be used. The first dosecan be given from about several months to about 5 to 7 weeks prior tofarrowing. The second dose of the vaccine then should be administeredsome weeks after the first dose, for example, about 2 to 4 weeks later,and vaccine can then be administered up to, but prior to, farrowing.Alternatively, the vaccine can be administered as a single 2 ml dose,for example, at about 5 to 7 weeks prior to farrowing. However, a 2 doseregimen is considered preferable for the most effective immunization ofthe baby pigs. Semi-annual revaccination is reconnected for breedinganimals. Boars may be revaccinated at any time. Also, sows can berevaccinated before breeding. Piglets born to unvaccinated sows may bevaccinated at about 3-10 days, again at 4-6 months and yearly orpreferably semi-annually thereafter.

The vaccine may also be combined with other vaccines for other diseasesto produce multivalent vaccines. It may also be combined with othermedicaments, for example, antibiotics. A pharmaceutically effectiveamount of the vaccine can be employed with a pharmaceutically acceptablecarrier or diluent to vaccinate animals such as swine, cattle, sheep,goats, and other mammals.

Other vaccines may be prepared according to methods well known to thoseskilled in the art as set forth, for example, in I. Tizard, AnIntroduction to Veterinary Immunology, 2nd ed. (1982), which isincorporated herein by reference.

As set forth above, commercial vaccine PRV's have been found to have thegI and gp63 genes deleted. Therefore gI and gp63 polypeptides producedby the methods of this invention can be used as diagnostic agents todistinguish between animals vaccinated with these commercial vaccinesand those infected with virulent virus.

To differentiate between infected and vaccinated animals, one couldemploy, for example, an ELISA assay. gI or gp63 protein, produced, forexample, in E. coli by recombinant DNA techniques (Rea, et al., supra.),is added to the wells of suitable plastic plates and allowed sufficienttime to absorb to the plastic (e.g., overnight, 20°-25° C.). The platesare washed and a blocking agent (e.g., BSA) is added to neutralize anyunreacted sites on the plastic surface. A wash follows and then the pigserum is added to the wells. After about 1 hour incubation at 20°-25°C., the wells are washed and a protein A-horseradish peroxidaseconjugate is added to each well for an about 1 hour incubation a 20°-25°C. Another wash follows and the enzyme substrate (o-phenylenediamine) isadded to the wells and the reaction is terminated with acid. Absorbencyis measured at 492 nanometers to quantitate the amount of gI or gp63antibody present in the serum. Lack of gI or gp63 antibody indicatesthat an animal is not infected. By testing for other PRV antibody, onecould establish whether or not a given animal was vaccinated. ##STR4##

We claim:
 1. A method for distinguishing an animal vaccinated with apseudorabies virus (PRV) vaccine lacking glycoprotein gI or gp63 from ananimal infected with a virulent wild-type pseudorabies virus withoutsacrificing the animal comprising: contacting serum from the animal withPRV glycoprotein gI or gp63, and detecting the presence or absence ofantibodies which specifically bind to PRV glycoprotein gI or gp63wherein the presence of antibodies indicates that the animal has beeninfected with a virulent wild-type pseudorabies virus.
 2. The method ofclaim 1 wherein the animal has been vaccinated with a PRV vaccinelacking glycoprotein gI and the serum is contacted with PRV glycoproteingI.
 3. The method of claim 1 wherein the presence or absence of theantibodies is detected using an ELISA.
 4. A mercantile kit useful inperforming the method of claim 1, comprising multiple containers whereinone of said containers has therein PRV glycoprotein gI or gp63 andanother of said containers has therein a reagent for detection ofantibodies which specifically bind to gI or gp63.
 5. The mercantile kitof claim 4 wherein the PRV glycoprotein is gI.